Uricase is an enzyme capable of decomposing uric acid by catalyzing the oxidation to allantoin and hydrogen peroxide. This enzyme plays an important role in the medical field, especially in biochemical diagnosis where it is used as a reagent for the detection of uric acid in serum or urine.
Uric acid is one of the principle products of the catabolism of purine bases and of the materials which they contain, such as nucleic acids. If such catabolism does not take place or if elimination of the uric acid thus produced does not occur, accumulations of these products in the blood or body tissue can be the cause of many disorders, especially gout, certain forms of rheumatism, certain calculi in the region of the urinary system and various tissue changes, especially in the cardio-vascular system. These disorders occur frequently because elimination of uric acid is rendered difficult by the very low solubility of this compound and increased concentrations of this compound due to any cause can bring about the formation of insoluble deposits.
Animal organs have heretofore been the principal source of uricase. Difficulties in extraction and purification of uricase from such sources have encouraged the development of uricase production from microorganisms. The production of uricase from various microorganisms, including bacteria, fungi and yeasts, is described in U.S. Pat. Nos. 3,431,176; 3,475,276; 3,620,923; 3,669,843; 3,767,533 and 3,810,820. It is likely that some trace of uricase might be found in any living organism but it cannot be predicted which organisms will yield uricase in sufficient quantities for any practical use.
U.S. Pat. Nos. 3,810,820 and 3,620,923 suggest that uricase may be obtained using bacteria of genus Micrococcus. However, no strians of bacteria of such genus have heretofore been identified as capable of providing useful levels of uricase in sufficient quantities to provide a commercially feasible source of uricase.
In the production of an enzyme such as uricase, whether by extraction from animal tissue or by fermentation of a microorganism, the desired enzyme is generally found in a liquid medium along with various other macromolecules such as proteins, including other enzymes, and/or other undersirable materials. Various methods have been used to purify the desired enzyme or separate it from at least some of the undesirable materials.
In purifying uricase, the enzyme, which is soluble in water but insoluble in organic solvents and insoluble in concentrated aqueous solutions of inorganic salts such as ammonium sulfate, can be recovered by precipitation either with an organic solvent which is miscible with water such as ethanol, methanol, isopropanol or acetone, or with a water soluble inorganic salt such as ammonium sulfate, mentioned previously. Salts or solvents can be removed by dialysis of a solution containing the redissolved precipitate.
Further purification can be accomplished, when necessary or desirable, by means of a series of precipitations from aqueous media, generally fractional precipitations, using organic liquids miscible with water or aqueous solutions containing ammonium sulfate. It is also possible to make use of adsorption upon hydroxyapatite, bentonite and alumina, and subsequent extraction, followed by elution using saline solutions. The purification can be carried still further by subjecting the thus treated products to chromatography, which may be a cyclic or noncyclic process, by making use of columns of substances which make it possible to eliminate those impurities, in particular, proteins, which are still present in the extract. The substances that have been suggested for this purpose include columns of cellulose ion exchange materials, dextrans and polyacrylamides. Elution may be effected by means of liquids in which there is a continuous or discontinuous change in the pH or in the molarity thereof.
Generally, even after such purification steps have been taken, the resulting uricase enzyme is not pure, but contains some amount of protein, e.g. other enzymes such as catalase. Catalase is a very undesirable interferant in a uricase preparation when the preparation is used for the quantitative analysis of uric acid in a peroxidase coupled detection system. The catalase catalyzes the decomposition of hydrogen peroxide and thus competes with the color forming reaction which is used to measure the quantity of uric acid present in a test solution. Prior to the present invention no purification methods were known to satisfactorily and efficiently separate catalase from uricase.